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, 288 (18), 12522-32

Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

Affiliations

Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

Waaqo Daddacha et al. J Biol Chem.

Abstract

HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.

Keywords: DNA Polymerase; DNA Replication; Enzyme Kinetics; Enzyme Mechanisms; Mutagenesis.

Figures

FIGURE 1.
FIGURE 1.
HIV-1 RT-mediated extension of DNA primers annealed to rNMP-containing templates. A 5′ end 32P-labeled DNA primer (10 nm) annealed to a 48-mer DNA template containing normal dAMP or rAMP at the 23rd position from the 3′ end (N: see Supplemental Table 1 for sequences) was extended with an equal activity of HIV-1 RT protein (green circle) for indicated time points. The reactions were conducted under macrophage dNTP pool (20–40 nm) (A) or activated T cell dNTP (2–5 μm) pools (B). C, primer extension on DNA templates containing rAMP at the 33rd position (lane 5–7) was conducted under macrophage dNTP pool for indicated duration. D, a 5′ end 32P-labeled 48-mer rAMP or dATP-containing DNA template annealed to the 23-mer DNA primer was incubated with HIV-1 RT, KOH, or none (C) for 60 min. The products were resolved by 14% denaturing PAGE and analyzed. F indicates fully extended product, P indicates an unextended primer, and CP indicates cleaved product. The arrow shows the pausing sites. The letter N in the T/P diagram shows the location of embedded rAMP (red) and corresponding dAMP (blue). The template sequences from the 3′ end of the annealed primer are marked at the sides of the gels. E, shown are circular dichroism results comparing a 22-mer primer annealed to 48-mer rNMP-free (black solid line) or rNMP-containing (red dashed line) template. The wavelength-showing discrepancy is marked as gray bars.
FIGURE 2.
FIGURE 2.
HIV-1 RT-mediated single round primer extension with rNTP-or dNTP-containing DNA template. A 5′ end 32P-labeled primer (8 nm) was annealed to 2-fold excess 48-mer DNA template with dAMP or rAMP and preincubated with HIV-1 RT. The primer extension was initiated by a mixture of macrophage (A and C) or activated T cell (B and D) dNTP concentrations and a molar excess of the unlabeled trap, poly(rA) annealed to oligo(dT). The reactions were terminated at 10, 20, and 60 min after the reaction initiation, T1, T2, and T3, respectively. Triplicate reactions were performed, and the products were quantified for macrophages (B) and activated T cells (D) dNTP pools. TC, HIV-1 RT was preincubated with a trap and labeled T/P mixture and the reaction was initiated by the addition of dNTPs. The reaction was terminated after 60 min for macrophage dNTP condition and after 20 min for T cell dNTP condition. No primer extension should be detected in this control. −T control, HIV-1 RT was preincubated only with the 32P-labled T/P in the absence of the trap, and the reaction was initiated by dNTPs and terminated at 20 min. In this condition, RT undergoes multiple rounds of primer extension and generates a greater amount of the fully extended product. C, control. As a negative control, no RT reaction was initiated by dNTP and terminated at 20 min. C, the reaction products were analyzed by 14% denaturing PAGE. F indicates fully extended product, P indicates unextended primer, and N indicates the location of rAMP and corresponding dAMP. The two pause sites are indicated by arrows. The template sequence from the 3′ end of the annealed primer was marked at the side of the gel.
FIGURE 3.
FIGURE 3.
HIV-1 RT-mediated matched and mismatched DNA primer extension with dAMP- or rNMP-containing DNA template. A 5′ end 32P-labeled matched or mismatched primer at its 3′ end was annealed to rAMP-containing or rAMP-free DNA template. This aligns rNMP at −1 position in HIV-1 RT active site. RT concentration that extends about 50% of the primers was determined and set as 1×. The primers were then extended with 1×, 0.5×, and 0.25× proteins for matched and 16×, 8×, or 4× for mismatched primers because the mismatch primer extension requires a higher RT amount to produce the detectable extended products. The reactions were then terminated after 5 min. A, the reaction products were analyzed by 14% denaturing PAGE. F indicates fully extended product, and P indicates unextended primer. The N in the diagram shows the location of embedded rNMP (rAMP) and corresponding dAMP (dAMP). B, triplicate reactions were performed, and products with 1× for matched and 16× for mismatched primers were quantified. The numbers above the graphs indicate -fold change as product on rNMP-containing template compared with that of dAMP-containing template.
FIGURE 4.
FIGURE 4.
SIV and AMV RT-mediated extension of DNA primer annealed to dAMP- or rNMP-containing DNA template. The same T/Ps containing dAMP or rAMP used in Fig. 1 was extended with an equal activity of SIVagm RT protein for indicated time points. The reactions were conducted under macrophage dNTP concentration for SIV RT (A) and T cells dNTP concentration for AMV RT (B). The products were resolved by 14% denaturing PAGE and analyzed. F indicates fully extended product, and P indicates unextended primer. The N in the diagram shows the location of embedded rAMP or dAMP.

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